BIOLOGY 3020 Fall 2008“Keys of Corn” Project

Plasmid isolation

Genetic Diversity in corn. Lots of different types of corn are offered for sale at the Pisac markets, in the Sacred Valley of the Incas.

Lecture Outline1. Laboratory Obejective2. Traditional Plasmid Prep3. Fast One-step lysis

method4. Estimation of Plasmid DNA

Concentration and purityA few weeks ago you identified a full length cDNA for a transcription factor from corn. The plasmid clones for these cDNAs have been ordered from the Arizona Genomics Institute (AGI). This week you will isolate these plasmids for further sequencing and cloning

2: Inoculate 5ml Broth culture and grow overnight

1: Primary Transformants and Single Colony Purification

Culture Clone for Plasmid Prep

Traditional Plasmid Prep1: Grow bacterial culture, spin down cells

2: Three step lysis procedure -a: Add lysozyme solution (degrade cell

wall)b: Add alkali - (punch holes in cell

membrane)c: Add Neutralization solution

3: Spin extract

4: Clean extract with phenol and chloroform (or column purification)

5: Precipitate DNA with alcohol

6: Spin down DNA, dry and resuspend in sterile water

100

1. Pellet 2 ml of fresh bacterial culture at maximum speed (at least 12,000 x g or 13,000 rpm) for 1 minute in the provided 2 ml Culture Tube.

2. Remove medium by decanting, taking care not to disturb bacterial pellet.

Fast Plasmid Prep Procedure (Eppendorf

Kit)

3. Add 400 µl of ICE-COLD Complete Lysis Solution. ONE step lysis is much faster

4. Mix thoroughly by constant vortexing at the highest setting for a full 30 seconds. This step is critical for obtaining maximum yield.

5. Incubate the lysate at room temperature for 3 minutes.

6. Transfer the lysate to a Spin Column Assembly by pipetting.

Fast Plasmid Prep Procedure

7. Centrifuge the Spin Column Assembly for 30–60 seconds at maximum speed.

8. Add 400 µl of Wash Buffer to the Spin Column Assembly.

9. Centrifuge the Spin Column Assembly for 30–60 seconds at maximum speed.

Fast Plasmid Prep Procedure

10. Remove the Spin Column from the centrifuge and decant the filtrate from the Spin Column Assembly Waste Tube. Place the Spin Column back into the Waste Tube and return it to the centrifuge.

12. Transfer the Spin Column into a Collection Tube - label with the provided code number (e.g. A1p2)

Fast Plasmid Prep Procedure

11. Centrifuge at maximum speed for 1 minute to dry the Spin Column.

13. Add 50 µl of Elution Buffer directly to the center of the Spin Column membrane and cap the the Collection Tube over the Spin Column.

Fast Plasmid Prep Procedure

14. Centrifuge at maximum speed for 30–60 seconds.

15. Remove and discard the Spin Column.

16. The eluted DNA can be used immediately for downstream applications or stored at -20°C.

Determine Plasmid DNA Concentration

Take 5 ul of plasmid DNA and add to 495 of water (100 fold dilution).

Use quartz cuvette to measure A260, A280, A230 and A320 in spectrophotometer (which has been blanked with water)

To determine DNA concentration:-Subtract A320 from A260 value. Multiply result by 50 µg/ml to find the concentration of DNA in the diluted sampleMultiply the result by the dilution factor (100 fold) and express the result in µg/µl and write on side of sample tube

1. Pellet 2 ml of fresh bacterial culture at maximum speed (at least 12,000 x g or 13,000 rpm) for 1 minute in the provided 2 ml Culture Tube.

2. Remove medium by decanting, taking care not to disturb bacterial pellet.

3. Add 400 µl of ICE-COLD Complete Lysis Solution.

4. Mix thoroughly by constant vortexing at the highest setting for a full 30 seconds. This step is critical for obtaining maximum yield.

5. Incubate the lysate at room temperature for 3 minutes.

6. Transfer the lysate to a Spin Column Assembly by pipetting.

7. Centrifuge the Spin Column Assembly for 30–60 seconds at maximum speed.

8. Add 400 µl of Wash Buffer to the Spin Column Assembly.

9. Centrifuge the Spin Column Assembly for 30–60 seconds at maximum speed.

Fast Plasmid Mini Prep - Step by Step

10. Remove the Spin Column from the centrifuge and decant the filtrate from the Spin Column Assembly Waste Tube. Place the Spin Column back into the Waste Tube and return it to the centrifuge.

11. Centrifuge at maximum speed for 1 minute to dry the Spin Column.

12. Transfer the Spin Column into a Collection Tube.

13. Add 50 µl of Elution Buffer directly to the center of the Spin Column membrane and cap the the Collection Tube over the Spin Column.

14. Centrifuge at maximum speed for 30–60 seconds.

15. Remove and discard the Spin Column.

16. The eluted DNA can be used immediately for downstream applications or stored at -20°C.

Fast Plasmid Mini Prep - Step by Step